RESEARCH Open Access
T ansposable elemen s in a ma ginal plan
popula ion: empo al luc ua ions p o ide
new insigh s in o genome e olu ion o wild
diploid whea
Alexande Belyaye
1*
, Ruslan Kalenda
2
, Leonid B odsky
1
, E ia a Ne o
1
, Alan H Schulman
2,3*
, Olga Raskina
1
Abs ac
Backg ound: How new o ms a ise in na u e has engaged e olu iona y biologis s since Da win’s seminal ea ise
on he o igin o species. T ansposable elemen s (TEs) may be among he mos impo an in e nal sou ces o
in aspeci ic a iabili y. Thus, we aimed o explo e he empo al dynamics o se e al TEs in indi idual geno ypes
om a small, ma ginal popula ion o Aegilops spel oides. A diploid c oss-pollina ed g ass species, i is a wild ela i e
o he a ious whea species known o hei la ge genome sizes con ibu ed by an ex ao dina y numbe o TEs,
pa icula ly long e minal epea (LTR) e o ansposons. The popula ion is cha ac e ized by high he e omo phy and
possesses a wide spec um o ch omosomal abno mali ies including supe nume a y ch omosomes, he e ozygosi y
o ansloca ions, and a iabili y in he ch omosomal posi ion o numbe o 45S and 5S ibosomal DNA ( DNA)
si es. We p opose ha a iabili y on he mo phological and ch omosomal le els may be linked o a iabili y a he
molecula le el and pa icula ly in TE p oli e a ion.
Resul s: Signi ican empo al luc ua ion in he copy numbe o TEs was de ec ed when p ocesses ha ake place
in small, ma ginal popula ions we e simula ed. I is known ha unde c i ical ex e nal condi ions, ou c ossing plan s
e y o en ansi o sel -pollina ion. Thus, h ee mo phologically di e en geno ypes wi h ch omosomal abe a ions
we e aken om a wild popula ion o Ae. spel oides, and he dynamics o he TE complex aced h ough h ee
ounds o sel ing. I was disco e ed ha : (i) a ious amilies o TEs a y emendously in copy numbe be ween
indi iduals om he same popula ion and he sel ed p ogenies; (ii) he luc ua ions in copy numbe a e TE- amily
speci ic; (iii) he e is a g ea di e ence in TE copy numbe expansion o con ac ion be ween game ophy es and
spo ophy es; and (i ) a small pe cen age o TEs ha inc ease in copy numbe can ac ually inse a no el loca ions
and could se e as a bona ide mu agen.
Conclusions: We hypo hesize ha TE dynamics could p omo e o in ensi y mo phological and ka yo ypical
changes, some o which may be po en ially impo an o he p ocess o mic oe olu ion, and allow species wi h
plas ic genomes o su i e as new o ms o e en species in imes o apid clima ic change.
Backg ound
Popula ions a e gene ally iewed as he elemen a y e o-
lu iona y uni [1,2]. A popula ion exis s as an in eg a ion
o indi iduals in ime and space ha can a y o e a se
o successi e gene a ions. Unde he in luence o spon-
aneous mu a ions, each popula ion becomes
he e ogeneous in i s gene ic s uc u e o e ime. Thus, a
popula ion comp ises a mix o di e en geno ypes e en
i i s indi iduals a e mo e o less pheno ypically simila .
Unde in ensi e p essu e om a pa icula (usually
en i onmen al) ac o , a common phenomenon in ma -
ginal popula ions, a shi in he geno ypic s uc u e o
he popula ion can occu . Such a shi may be ega ded
as he elemen a y e en o mic oe olu ion.
T ansposable elemen s (TEs) may be among he mos
impo an in e nal sou ces o geno ypic popula ion
* Co espondence: [email p o ec ed]ai a.ac.il; [email p o ec ed]
1
Ins i u e o E olu ion, Uni e si y o Hai a, Moun Ca mel, Hai a, Is ael
2
MTT/BI Plan Genomics Labo a o y, Ins i u e o Bio echnology, Viikki
Biocen e , Uni e si y o Helsinki, Helsinki, Finland
Belyaye e al.Mobile DNA 2010, 1:6
h p://www.mobilednajou nal.com/con en /1/1/6
© 2010 Belyaye e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
change as a esul o hei abili y o c ea e mu a ions,
al e gene exp ession, and p omo e ch omosomal abe -
a ions [3-5]. These ancien , ubiqui ous, and dynamic
componen s o euka yo ic genomes comp ise up o 80%
o he la ge genomes o ce eals [6,7]. Da a accumula ed
o e he las 30 yea s sugges ha TEs can ha e majo
e ec s on genome o ganiza ion and unc ion [4,6,8,9].
Ne e heless, i is s ill unclea whe he and o wha
deg ee he TEs con ibu e o e olu iona ily signi ican
shi s in he geno ypic s uc u e o popula ions. Popula-
ion gene ic heo y assumes ha he dynamics o ans-
posable elemen s in na u al popula ions e lec a balance
be ween he endency o hese elemen s o inc ease
h ough ansposi ion and hei emo al h ough na u al
selec ion ac ing agains indi iduals wi h a high elemen
copy numbe [10,11]. Se e al s udies om di e en bio-
logical sys ems indica e ha he pa e ns o a single TE
amily can a y in aspeci ically [12-15] and empo ally
[16]. Examina ion o he in aspeci ic dynamics o TEs,
especially in ma ginal popula ions whe e mic oe olu-
iona y p ocesses a e in ensi ied [2], could conside ably
con ibu e o he unde s anding o key biological e en s
such as specia ion.
One o he mos e ec i e ways o unde s and he
dynamics o he TEs o e ime and space may be an
ecological app oach, whe eby se e al gene a ions o
plan s om na u al popula ions a e in es iga ed. He e,
we ha e chosen a small, ma ginal and h ea ened popu-
la ion o Aegilops spel oides (2n = 2× = 14) as a model
o explo a ion o he dynamics o se e al TE amilies
belonging o Classes I and II ( espec i ely mo ing ia
RNA and DNA in e media es). The species is a wild
ela i e o he a ious whea species known o hei
la ge genome sizes [17,18] con ibu ed o by an ex ao -
dina y numbe o TEs, pa icula ly long e minal epea
(LTR) e o ansposons. Ae. spel oides was p oposed o
be he closes o he wild diploid p ogeni o o he G
genomes and B genomes o polyploid whea [19-21].
The s udy popula ion is loca ed on he wes e n banks o
he Kishon Ri e (Hai a Bay a ea, Is ael). The popula ion
is cha ac e ized by high he e omo phy and possesses a
wide spec um o ch omosomal abno mali ies, including
supe nume a y ch omosomes, he e ozygosi y o ans-
loca ions, and a iabili y in he ch omosomal posi ion o
numbe o 45S and 5S ibosomal DNA ( DNA) si es
[22,23]. We p opose ha a iabili y in mo phological
and ch omosomal le els may poin o a iabili y a he
molecula le el and pa icula ly in TE p oli e a ion.
Thus we aimed o de ec and e alua e di e ences in TE
copy numbe s be ween indi iduals om he Kishon
popula ion and o ace inhe i ance o hese de ia ions
in successi e gene a ions. The b oade goal o he s udy
was o examine whe he TE dynamics could be asso-
cia ed wi h mo phological o ka yo ypical changes, some
o which a e po en ially impo an o he e olu iona y
p ocess.
Resul s
Expe imen al design
Ae. spel oides is p edominan ly a c oss-pollina ed bu
sel -compa ible species [24]. Th ee o iginal plan s we e
selec ed om a small (app oxima ely 100 m
2
), ma ginal,
deg ade popula ion o Ae. spel oides (see Me hods).
Each selec ed o iginal geno ype ep esen s h ee g oups
o p e iously in es iga ed plan s ( i e o se en indi idual
o iginal spikes in each g oup), which ha e been clus-
e ed due o hei mo phological and cy ogene ic simi-
la i y, including spike mo phology, B ch omosome
exis ence, appea ance o addi ional 5S DNA ch omoso-
mal clus e s, and speci ic ch omosomal ea angemen s
[22,23].
The p ogeny om each geno ype we e ob ained in
h ee ounds o sel ing. We simula ed he si ua ion in
na u e whe e, in ma ginal popula ions unde c i ical
ex e nal condi ions, ou c ossing plan s and pa icula ly
Ae. spel oides e y o en ansi o sel -pollina ion
[24-26]. The copy numbe s o se e alTE amilieswe e
de e mined by quan i a i e polyme ase chain eac ion
(qPCR) o each o iginal geno ype and i s o sp ing.
qPCR da a we e e i ied by do blo analysis. The ans-
posi ional ac i i ies o TEs we e in e ed by in e - e o-
ansposon ampli ied polymo phism (IRAP)
e o ansposon display [27]. Gene a ional changes in
he copy numbe o TE amilies we e compa ed wi h
hose o he non-mobile, highly epe i i e andem epea
Spel 52 and he 5S ibosomal RNA ( RNA) genes
( DNA).
Dynamics o TE in successi e gene a ions
The copy numbe s o TE amilies ha we in es iga ed
a e p esen ed in Addi ional ile 1 and in g aphic o m in
Figu e 1a. The changes in TE copy numbe in a se o
successi e gene a ions was measu ed by wo di e en
me hods: qPCR and do blo . These p oduced consis en
esul s.
The i s salien poin is ha gene a i e issues, in mos
cases, possess highe numbe s o TEs han do ege a i e
issues. This obse a ion is ue o mos TEs excep
hose ela ed o he A hila amily (Sab ina and Wham).
Fo example, he numbe o WIS2 e o ansposons in
he spikes o G13 S1 exceeded ha in lea es by 59%, o
app oxima ely 18,000 copies. The copy numbe s o bo h
he ibe-speci ic Spel 52 andem epea s and he 5S
RNA genes we e also gene ally highe in spikes han in
lea es (Addi ional ile 1, Figu e 1a).
The second ema kable ea u e is ha he copy num-
be s o all in es iga ed TEs a ied signi ican ly o e
h ee successi e gene a ions, and ha each geno ype
possessed indi idual TE dynamics o e his pe iod. The
Belyaye e al.Mobile DNA 2010, 1:6
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TE copy numbe s dec eased o inc eased signi ican ly,
wi h a ise in one gene a ion ollowed by a d op in he
nex and ice e sa in an oscilla o y ashion. In gene a-
i e issues, hese oscilla ions displayed much highe
ampli udes. Pa icula TEs dec eased up o 23% om
hei ini ial quan i y, as in he case o Daniela e o an-
sposons in G9 spikes whe e he copy numbe o his
elemen d opped om app oxima ely 62,000 copies in
S1 o app oxima ely 15,000 copies in S2 (Addi ional ile
1, Figu e 1a). We could no ace u he TE dynamics
in his geno ype since he S2 gene a ion was s e ile. The
la ges absolu e ise in TE numbe was documen ed also
o Daniela in G13 spikes whe e he copy numbe ose
om app oxima ely 18,000 copies in S2 o app oxi-
ma ely 52,000 copies in S3. The la ges ela i e ise in
TE abundance in one gene a ion was seen o he Sab-
ina elemen , whe e in G14 i s numbe in spikes
inc eased by 672% in S3 as compa ed o S2. A simila
jump in his geno ype be ween S2 and S3 was obse ed
also o WIS2,Wilma, and Wham.
Geno ypes 9 and 13 we e dis inc by he ampli udes in
hei numbe s o copies o pa icula TE amilies o e
he gene a ions analyzed, bu he endencies we e he
same. Common ends included a signi ican inc ease o
he highly abundan e o ansposons, WIS2,Daniela,
and Fa ima, in he gene a i e issues o he S1
Figu e 1 Copy numbe s and mo phology.(a) Dynamics o ansposable elemen (TE) copy numbe s in h ee sel -pollina ed gene a ions o
h ee geno ypes om he Kishon popula ion o Aegilops spel oides (shown by lines). TE copy numbe s o he TS-84 popula ion we e used as
con ols. TE copy numbe s o a ailable sibs in sel ing gene a ions a e shown by sepa a e do s. (b) Changes in spike mo phology in h ee sel -
pollina ed gene a ions o h ee geno ypes om he Kishon popula ion o Ae. spel oides. Spike mo phology o plan s om he TS-84 popula ion
was used as he con ol.
Belyaye e al.Mobile DNA 2010, 1:6
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gene a ion, ollowed by a simila dec ease in hese ele-
men s in he S2 gene a ion (Addi ional ile 1, Figu e 1a).
En/Spm-like ansposons, Cassand a e o ansposons,
and 5S RNA genes exhibi ed simila dynamics in hese
geno ypes. Wilma e o ansposons did no change in
he S1 gene a ion bu hen inc eased in numbe in S2.
The copy numbe o Sukkula elemen s did no change
in he S1 gene a ion, bu hen d opped in S2. Sab ina
and Wham elemen s emained app oxima ely on he
same le el as in he o iginal plan s. The TE dynamics in
ege a i e issues we e app oxima ely he same as in
gene a i e issues bu wi h less ampli ude. Fo echnical
easons, we do no ha e measu emen s o he copy
numbe o Spel 52 in he spikes o he o iginal plan s
o G9, bu he es o he measu emen s showed a en-
dency o his andem epea o dec ease in abundance
in gene a i e and ege a i e issues o e he gene a ions
examined.
Geno ype 14 di e ed in TE copy numbe empo al
dynamics om he wo p e iously desc ibed geno ypes.
Cy ogene ic da a (see below) make i possible o p opose
ha his geno ype was al eady sel -pollina ed o a leas
one gene a ion. WIS2, Wilma, Sab ina, Wham, and Suk-
kula elemen s demons a ed signi ican , successi e
dec eases in copy numbe s in S1 and S2 ha we e hen
ollowed by inc eases in S3. Daniela, En/Spm,andCas-
sand a elemen s inc eased in copy numbe in S1 and
successi ely dec eased in S2 and S3. Fa ima e o an-
sposons exhibi ed copy numbe dynamics in G14 simila
o hose seen in G9 and G13: inc eased abundance in
S1, dec easing in S2, and again inc easing in S3. In G14,
oo, he TE dynamics in ege a i e issues we e app oxi-
ma ely he same as o gene a i e issues, bu wi h less
ampli ude. The numbe o Spel 52 epea s, bo h in
spikes and lea es, dec eased in S1, inc eased in S2, and
dec eased in S3. In spi e o a signi ican inc ease o copy
numbe in S2, he o e all endency was o educ ion.
The 5S RNA genes successi ely inc eased in copy num-
be in S1 and S2, and hen dec eased in S3.
To explo e seg ega ion in he p ogenies o a single
geno ype, TE copy numbe s in sibs om G9 and G13
geno ypes we e de e mined (Table S1 in Addi ional ile
2). Signi ican a ia ions o e h ee gene a ions o wo
geno ypes we e obse ed. The ampli ude o TE abun-
dance in sibs could no be explained by simple ch omo-
some seg ega ion (see below) and, he e o e, poin s o
mobile elemen ac i i y.
A TE display me hod such as IRAP [27] would be
expec ed o show polymo phisms consonan wi h la ge
changes in TE copy numbe . He e, IRAP analyses we e
conduc ed on DNA om spikes a he mic ospo ogen-
esis s age, and showed a high le el o polymo phism
om gene a ion o gene a ion (Figu e 2). G13 showed
he mos unique bands, wi h e o ansposons WIS2 and
Daniela p oducing he g ea es numbe .
Va ious o ms o ecombina ion may, o cou se, b ing
TEs su icien ly close ha a new IRAP band would
appea in he absence o a new in eg a ion e en . Fo
any gi en IRAP polymo phism, i s o igin as an in eg a-
ion e en can be explici ly p o en only by iden i ying
an emp y si e, om a plan line missing he IRAP p o-
duc , o one o he wo TEs ha oge he se ed as he
empla e. To ind e idence o indi idual ans o ma ion
e en s, we cloned and sequenced 20 unique IRAP bands
(Figu e 3). A leas hal o he new inse ion si es we e
in epe i i e DNA, and he e o e unsui ed o iden i ying
he o iginal emp y si es. O he 20, (Table S2 in Addi-
ional ile 2), 4 o hem (a Daniela,aWis2,and2Suk-
kula inse ions) we e in non- epe i i e si es ha could
be iden i ied in a sequence da abase. The ad en o new,
unique, ampli ied IRAP bands ha esul ed om new
TE inse ions con i ms he exis ence o ansposi ionally
ac i e TEs in he h ee geno ypes o Ae. spel oides.
Mul i a ia e analyses
The da a dis ibu ion in p inciple componen analysis
(PCA) (Figu e 4a) demons a es he clea sepa a ion o
lea es and spikes o each gene a ion, no malized pai -
wise o each TE and non-TE epea in con ol plan s.
The sepa a ion is along he i s p incipal componen
(PC1), which co e s 62% o ini ial da a a iabili y. The
PC1 (Figu e 4b) con as s a g oup o TEs (Sukkula,
Fa ima, Cassand a, En/Spm, and Daniela) and wo non-
TE sequences (Spel 52 and 5S DNA) e sus ano he
g oup o TEs (Wilma, Sab ina, Wham,andWIS2).
The e o e, he pa e ns o ela i e gene a ional copy
numbe a ia ion o hese wo g oups o TEs ega ding
hei numbe s in con ol plan s a e di e en in spikes
and lea es. The i s g oup o TEs and bo h non-TE
sequences show la ge luc ua ions in lea es o e suc-
cessi e gene a ions, and he TEs o he second g oup
show la ge changes in spikes.
The second p incipal componen (PC2) ep esen s he
a iabili y ha is independen o he lea -spike con as
(Figu e 4b). When his componen is u he dissec ed
by ype o epea (Figu e 4c), he issue-independen
a iance shows opposi e ends o TE and non-TE
epea s. This dicho omous end holds ac oss gene a-
ions, geno ypes, and issues. We u he in es iga ed
he na u e o his end o all nine TE amilies. Bo h
aw copy numbe s and hose no malized by he con ol
da a we e hus no malized by he numbe o one o he
non-TE sequences (5S DNA) and by con ol plan s
(TS-84) (Figu e 4d). Fo bo h ypes o da a no maliza-
ion, he cen alized pa e ns o change in copy numbe
o he TEs and o Spel 52 o e he h ee geno ypes
and ac oss issues and gene a ions demons a e ha : (i)
he copy numbe luc ua ion o non-TE sequence Spel
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52 wi h espec o 5S DNA is insigni ican ac oss is-
sues and gene a ions (b oken ed line) while luc ua ions
o TE sequences a e high; (ii) he in e gene a ional pa -
e n o copy numbe changes o all TEs is highly co e-
la ed; (iii) he e is li le ela i e TE copy numbe
a ia ion in lea es hough i is e y s ong in spikes. I
shows a minimum in he S2 gene a ion, and a maxi-
mum in he S3 gene a ion. In sum, he sepa a ion o TE
and non-TE sequences in PC2 appea s ela ed o hei
di e en pa e ns o copy numbe dynamics ac oss gen-
e a ions in spikes.
De ia ions o eal copy numbe s in lea es and spikes
om he s ochas ic model
The o iginal plan s o c oss-pollina ed Ae. spel oides
a e he e ozygous o epe i i e DNA sequences, ch o-
mosomal pa e ns, and ch omosomal ea angemen s
(Figu es 5 and 6). In o de o sepa a e he ole o he
p e-exis ing he e ozygosi y on he TE copy numbe in
Figu e 2 In e - e o ansposon ampli ied polymo phism (IRAP) analyses o se e al ansposable elemen s (TEs) in he p ogeny o
h ee geno ypes. Unique bands ha appea in S2 and a e inhe i ed in S3 a e shown by ed a ows. An example o he e ozygosi y displayed in
he IRAP pa e n is shown in he blue squa e. An example o band loss in S2 and S3 is shown in he g een squa e. An example o band
appea ance in S2 and S3 is shown in he yellow squa e.
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he o iginal plan s om o he mechanisms ha can
change TE amoun s in successi e gene a ions, such as
ansposi ion, excision, o ecombina ional elimina ion,
we c ea ed a ‘naï e’model in which he seg ega ion o
pa en al ch omosomes in sel ed p ogenies was simu-
la ed. The null hypo hesis implies ha : (i) c oss-polli-
na ed pa en al plan s we e ini ially he e ozygous o
he con en o TEs in he homologues; (ii) only he
andom seg ega ion o homologues in meiosis du ing
male and emale game ogenesis leads o a change in
TE copy numbe in inb ed o sp ing. Fo modeling we
ook only he G13 geno ype as he geno ype wi h a
s able ch omosome numbe , and only he dynamics o
Class I elemen s we e simula ed.
We ca ied ou 100 ealiza ions o s ochas ic ime
cou se simula ions o copy numbe s in spikes and lea es
ac oss en gene a ions. Thus, he copy numbe s in lea es
and spikes we e calcula ed in 100 simula ed siblings o
each gene a ion o plan s, and hese 100 simula ed siblings
om each o he i s h ee plan gene a ions o m he
gene a ion-speci ic basic dis ibu ions o copy numbe s in
lea es and spikes. The de ia ions o copy numbe s in eal
siblings om he basic simula ed dis ibu ions, sepa a ely
in lea es and spikes, es he null hypo hesis.
The signi icance o de ia ions in each gene a ion was
calcula ed by a - es and he non-pa ame ic Mann-
Whi ney U es (Table S7 in Addi ional ile 2). The sig-
ni icance esul s o wo es s a e in good mu ual conco -
dance. Taking his in o conside a ion, he F- es
s a is ics we e used o signi icance es ima ion o he
cumula i e de ia ion in posi i e o nega i e di ec ions o
eal da a om simula ed siblings ac oss h ee gene a-
ions o each ype o TE sepa a ely. The F s a is ic is:
FW Wmax min
/
whe e W
max
and W
min
a e ei he W
p
o W
n
depending
on which one is la ge . The s a is ic W
p
is de ined as he
sum o squa es ei he o eal - es alues, i - es is
Figu e 3 Analysis o unique in e - e o ansposon ampli ied polymo phism (IRAP) bands.In hisexample,IRAPwasconduc edwi h
p ime 2109, ma ching he Daniela e o ansposon long e minal epea (LTR) (Table S2 in Addi ional ile 2). The band was cu and sequenced.
Two new p ime s we e designed o ma ch he sequence and he uniqueness o he band was checked on he se o DNA samples. This new
inse ion is in epe i i e DNA (Table S2 in Addi ional ile 2).
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posi i e, o al e na i e expec ed posi i e - es s (a small
alue o Q25 le quan ile o - es alue dis ibu ion), i
he eal - es is nega i e:
W P a P a P a
p es es es
1
2
2
2
3
2
(,) (, ) (, )
Simila ly:
W N a N a N a
n es es es
1
2
2
2
3
2
(,) (, ) (, )
S a is ics W
p
and W
n
a e chi-squa e dis ibu ed wi h
deg ees o eedom equaling h ee each. Thus, he F- es
has (3,3) deg ees o eedom. The es shows how co o-
bo a i e - es di ec ions and alues a e ac oss h ee
plan gene a ions o he pa icula TE ype. The + and
-logP alues o his F- es s a is ic ac oss TE ypes a e
showninFigu e4e.Thesigno helogP alue e lec s
he o ing o - es s ei he up (W
max
=W
p
;W
min
=W
n
)
o down (W
max
=W
n
;W
min
=W
p
) omsimula ions.
Thus, long down ba s indica e signi ican cumula i e
de ia ion o eal da a down om simula ions ac oss
h ee plan gene a ions.
The g aph (Figu e 4e) shows ha copy numbe s o all
TE ypes besides Daniela in lea es o G13 we e signi i-
can ly down egula ed in ela ion o he simula ions, bu
his is no ue o spikes. The explana ion could be ha
a high TE copy numbe in he male game es o he o i-
ginal plan in e s a gene ally high simula ed le el o
copy numbe s in spikes and lea es. Howe e , a plan
could su i e only i he TE copy numbe is ela i ely
low in diploid cells, pe mi ing, howe e , a high copy
numbe in game es. Thus, plan s wi h high copy num-
be s, bo h in game es and diploid cells, will be elimi-
na ed om he popula ion, p o iding he obse ed
di e ence be ween eal copy numbe s in game es and in
diploid cells o su i o s.
Mo phological and ka yo ypical changes in successi e
gene a ions
Ae. spel oides is a dimo phic species appea ing in na -
u al popula ions as wo mo pho ypes o subspecies;Ae.
spel oides ssp. ligus ica and Ae. spel oides ssp. auche i.
The auche i ype is cha ac e ized by cylind ical spikes
wi h widely in e spe sed spikele s. The apical spikele is
awned, and he achis is ough. The ligus ica ype is
cha ac e ized by dense , wo- owed ea s in which he
la e al spikele s a e also awned and he achis is b i le
(Figu e 1b). Signi ican mo phological and ka yo ypic
abno mali ies in successi e gene a ions we e obse ed,
pa icula ly: educ ion o spike awns, appea ance o
in e media e ligus ica-auche i pheno ypes, abno mal
quan i y o spikele s and lo e s, and spike size educ ion
(Figu e 1b).
Figu e 4 P inciple componen analysis (PCA).(a) PCA analysis: spikes e sus lea es o ansposable elemen (TE) copy numbe changes o e
he gene a ions, no malized by he TS-84 con ol. (b) PCA analysis: wo g oups o TE, spike ac i e and lea ac i e. (c) Second p incipal
componen (PC2)-based o de o TE ac i a ion. (d) No malized and cen alized pa e ns o he h ee geno ypes o Spel 52 and TEs ac oss issues
and gene a ions. (e) De ia ions o eal copy numbe s in lea es and spikes om he s ochas ic model.
Belyaye e al.Mobile DNA 2010, 1:6
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Fo ka yo ypic analysis we used se e al ch omosomal
ma ke s in luo escen in si u hyb idiza ion (FISH)
expe imen s: 5S DNA and 45S DNA p obes, ce eal
cen ome e-speci ic sequence 1 (CCS-1) [28], species-
speci ic Spel 1 [29] and ibe-speci ic Spel 52 [30] an-
dem epea s. These p obes we e used o ch omosome
iden i ica ion and localiza ion o ch omosomal ea an-
gemen s. The genomic o mulas o he in es iga ed
plan s we e as ollows: G9 (2n = 14 + 3B), G9 S1 o S2
(2n = 14 + 5B), G13, S1 o S3 (2n = 14), G14 (2n = 14
+ 2B), G14 S1 (2n = 14 + 3B), G14 S2 (2n = 14 + 4B),
and G14 S3 (2n = 14 + 3B) (Figu es 5 and 6).
Dynamics o Spel 1, Spel 52, and 5S DNA clus e s in
successi e gene a ions e ealed by FISH
Cy ogene ical analysis e ealed d ama ic di e ences
be ween genome o ganiza ions o plan s om he
Kishon popula ion in compa ison wi h TS-84 plan s
ep esen ing he cen e o he Ae. spel oides dis ibu ion.
Fi s , Kishon plan s los a majo i y o species-speci ic
andem epea Spel 1, which appea s usually as a com-
ponen o e minal adenine and hymine (AT) - ich he -
e och oma ic blocks in bo h a ms o almos all
ch omosomes in TS-84 (Figu e 6d). Only one o wo
e minal clus e s o Spel 1 pe diploid genome we e
e ealed in all s udied geno ypes, which means ha up
o 97% o he clus e s we e elimina ed (Figu e 5b, c, e
and Figu e 6c, Table 1). We obse ed a simila pic u e
o Spel 52 dis al clus e s: 57% o 82% we e elimina ed
in geno ypes om he Kishon popula ion (Figu es 5 and
6, Table 1). Thus, he amoun o Spel 1 is 13 imes
lowe and Spel 52 1.5 imes lowe han in he con ol
geno ype TS-84 om he cen e o he species
Figu e 5 Fluo escence in si u hyb idiza ion (FISH) and di e en ial s aining wi h 4’,6-diamidino-2-phenylindole (DAPI) on soma ic and
meio ic ch omosomes o Aegilops spel oides (pa 1).(a) FISH wi h 5S DNA, 45S DNA and s aining wi h DAPI on soma ic ch omosomes o
he o iginal G9 plan (le ). Ch omosomes 1, 6 (a ows), and B ch omosomes ca y addi ional 5S DNA si es ( igh ). (b) FISH wi h Spel 52, Spel 1
(a ows on B ch omosomes), 5S DNA and 45S DNA on he soma ic ch omosomes o he G9 S1 plan (le ); FISH wi h 5S DNA ( igh ).
Ch omosomes 1 and 6 ca y addi ional 5S DNA si es (a owed). (c) F om le o igh : FISH wi h 5S DNA and 45S DNA, and DAPI on he
meio ic ch omosomes o he G9 S2 plan ; 5S DNA p obe alone, ch omosomes 1, 6 (a ows), and B ch omosomes ca y addi ional 5S DNA si es;
FISH wi h Spel 52 and Spel 1 on he same ch omosomes; FISH wi h CCS-1 and 45S DNA (a pe icen ic in e sion is a owed); he scheme o he
main ch omosomal ea angemen s (see legend). (d) DAPI (le ) and FISH (middle) wi h 5S DNA and 45S DNA on he meio ic ch omosomes o
he G13 S2 plan . A scheme o he main ch omosomal ea angemen s ( igh ). (e) FISH wi h Spel 52 and Spel 1 on he meio ic ch omosomes o
he G13 S3 plan (le ). Small Spel 52 clus e (a ow) ma ks pa acen ic in e sion in he long a m o he ch omosome 5. FISH wi h 5S DNA and
45S DNA wi h DAPI s aining (middle). Bo h e mini o ch omosome 5 a e in ol ed in he e ologous synapses (whi e a ows); he e ozygous
dele ion on he ch omosome 6 is shown by yellow a ow. Ch omosomes 1 and 6 ca y addi ional 5S DNA clus e s (a ows in igh ). The DNA
p obes and s aining: (a-e) 5S DNA, Spel 52 and ce eal cen ome e-speci ic sequence 1 (CCS-1) in ed; 45S DNA and Spel 1 in g een; di e en ial
s aining wi h DAPI in blue; (b) 5S DNA (yellow) and 45S DNA (blue) in pseudocolo s.
Belyaye e al.Mobile DNA 2010, 1:6
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dis ibu ion. These da a show ha in he ma ginal
Kishon popula ion a high a io o ecombina ion occu s
in e minal and dis al ch omosomal egions. E iden ly,
e minal clus e s o Spel 1 a e he p ima y a ge s o
dele ions, ollowed by dis ally loca ed Spel 52 clus e s.
To he ex en ha ch omosome e mini ha e a simila
s uc u e (AT- ich he e och oma ic bands en iched wi h
clus e s o Spel 1 and/o Spel 52 andem epea s and
TEs), hey could be in ol ed no only in homologous
bu also in he e ologous ecombina ion ollowed by
ch omosomal ea angemen s. In he Kishon popula ion
we obse ed an i e e sible elimina ion o e minal, spe-
cies-speci ic Spel 1 andem epea s and a signi ican
educ ion o he dis al, ibe-speci ic andem epea
Spel 52 as a esul o high a es o homologous and
he e ologous ecombina ion in his small inb ed
popula ion.
FISH expe imen s e ealed ha a signi ican inc ease
o dec ease in copy numbe migh happen wi hou
change in he clus e quan i y. Fo example, we aced a
signi ican educ ion in Spel 52 copy numbe om he
G9 o iginal plan o G9 S2 (Figu e 1a), while clus e
numbe s inc ease om 9 o 11 in S1 and d op again o
9inS2(Table1).Inspikeso G14S29,200copieso
Spel 52 a e sha ed be ween 9 clus e s while in S3 only
3,800 copies a e assigned o 10 ch omosomal clus e s
(Table 1 and Addi ional ile 1). The dynamics o Spel
52 clus e s in G14 a e as ollows: he G14 o iginal line,
10 clus e s; G14 S1 o S8; G14 S2 o S9; and G14 S3 o
S10 clus e s pe diploid genome. Howe e , he ch omo-
somal dis ibu ion o Spel 52 clus e s signi ican ly di -
e s be ween hese geno ypes. Thus, bo h homologues o
ch omosome 6 ca y Spel 52 clus e s in he long a ms
o he o iginal (Figu e 6a) and S1 plan s, while no clus-
e s we e de ec ed ( ha is, all clus e s ha e been
dele ed) as ea ly as in S1 (no shown), S2, and S3 (Fig-
u e 6a and 6b). One homologue o ch omosome 5 in
he G14 o iginal plan ca ies wo clus e s in he he e o-
zygo e in he long a m, whe e he second clus e is a
esul o ansloca ion (Figu e 6a). In S1 and S2 only
one clus e s ill exis s in he he e ozygo e, which p o-
ides e idence o dele ion o a second clus e as a
Figu e 6 Fluo escence in si u hyb idiza ion (FISH) and di e en ial s aining wi h 4’,6-diamidino-2-phenylindole (DAPI) on soma ic and
meio ic ch omosomes o Aegilops spel oides (pa 2).(a) F om le o igh : DAPI and FISH wi h he CCS-1 and 45S DNA on he meio ic
ch omosomes o he o iginal G14 plan ; FISH wi h Spel 52 and Spel 1. The clus e s o Spel 1 ha ma k a homozygous pa acen ic in e sion on
ch omosome 4, and a Spel 1 clus e on he B ch omosome a e a owed. FISH wi h 5S DNA and 45S DNA on he same ch omosomes. A
scheme o he main ch omosomal ea angemen s. (b) FISH wi h Spel 52 and Spel 1 on he meio ic ch omosomes o he G14 S2 plan (le ).
The clus e s o Spel 1 ha ma k a homozygous pa acen ic in e sion on ch omosome 4 and clus e o Spel 52 ha ma ks a he e ozygous
in e sion on he ch omosome 5 a e a owed. FISH wi h 5S DNA and 45S DNA (middle). B ch omosomes ca y 5S DNA clus e s in bo h a ms.
The scheme o he main ch omosomal ea angemen s ( igh ). (c) FISH wi h Spel 52 and Spel 1 on he meio ic ch omosomes o he G14 S3
plan (le ). Homozygous pa acen ic in e sion on ch omosome 4 is a owed. FISH wi h 5S DNA and 45S DNA on he same ch omosomes
( igh ). (d) Soma ic ch omosomes o TS 84, s aining wi h DAPI (le ). FISH wi h Spel 52 and Spel 1 on he same ch omosomes ( igh ). The DNA
p obes and s aining: (a-d) 5S DNA, Spel 52 and ce eal cen ome e-speci ic sequence 1 (CCS-1) in ed; 45S DNA and Spel 1 in g een; di e en ial
s aining wi h DAPI in blue; (a) 5S DNA (yellow) and 45S DNA (blue) in pseudocolo s.
Belyaye e al.Mobile DNA 2010, 1:6
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doi:10.1186/1759-8753-1-6
Ci e his a icle as: Belyaye e al.: T ansposable elemen s in a ma ginal
plan popula ion: empo al luc ua ions p o ide
new insigh s in o genome e olu ion o wild diploid whea . Mobile DNA
2010 1:6.
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